Marc Staal

Temperature excludes N2-fixing heterocystous cyanobacteria in the tropical oceans

Whereas the non-heterocystous cyanobacteria Trichodesmium spp. are the dominant N2-fixing organisms in the tropical oceans, heterocystous species dominate N2 fixation in freshwater lakes and brackish environments such as the Baltic Sea. So far no satisfactory explanation for the absence of heterocystous cyanobacteria in the pelagic of the tropical oceans has been given, even though heterocysts would seem to represent an ideal strategy for protecting nitrogenase from being inactivated by O2, thereby enabling cyanobacteria to fix N2 and to perform photosynthesis simultaneously. Trichodesmium is capable of N2 fixation, apparently without needing to differentiate heterocysts. Here we show that differences in the temperature dependence of O2 flux, respiration and N2 fixation activity explain how Trichodesmium performs better than heterocystous species at higher temperatures. Our results also explain why Trichodesmium is not successful in temperate or cold seas. The absence of heterocystous cyanobacteria in the pelagic zone of temperate and cold seas, however, requires another explanation.

Modeling the dynamic regulation of nitrogen fixation in the cyanobacterium Trichodesmium sp.

ABSTRACT A physiological, unbalanced model is presented that explicitly describes growth of the marine cyanobacterium Trichodesmium sp. at the expense of N2 (diazotrophy). The model involves the dynamics of intracellular reserves of carbon and nitrogen and allows the uncoupling of the metabolism of these elements. The results show the transient dynamics of N2 fixation when combined nitrogen (NO3−, NH4+) is available and the increased rate of N2 fixation when combined nitrogen is insufficient to cover the demand. The daily N2 fixation pattern that emerges from the model agrees with measurements of rates of nitrogenase activity in laboratory cultures of Trichodesmium sp. Model simulations explored the influence of irradiance levels and the length of the light period on fixation activity and cellular carbon and nitrogen stoichiometry. Changes in the cellular C/N ratio resulted from allocations of carbon to different cell compartments as demanded by the growth of the organism. The model shows that carbon availability is a simple and efficient mechanism to regulate the balance of carbon and nitrogen fixed (C/N ratio) in filaments of cells. The lowest C/N ratios were obtained when the light regime closely matched nitrogenase dynamics.

Ultrabright planar optodes for luminescence life-time based microscopic imaging of O2 dynamics in biofilms

New transparent optodes for life-time based microscopic imaging of O₂ were developed by spin-coating a μm-thin layer of a highly luminescent cyclometalated iridium(III) coumarin complex in polystyrene onto glass cover slips. Compared to similar thin-film O₂ optodes based on a ruthenium(II) polypyridyl complex or a platinum(II) porphyrin, the new planar sensors have i) higher brightness allowing for much shorter exposure times and thus higher time resolution, ii) more homogeneous and smaller pixel to pixel variation over the sensor area resulting in less noisy O₂ images, and iii) a lower temperature dependency simplifying calibration procedures. We used the new optodes for microscopic imaging of the spatio-temporal O₂ dynamics at the base of heterotrophic biofilms in combination with confocal imaging of bacterial biomass and biofilm structure. This allowed us to directly link biomass distribution to O₂ distribution under both steady state and non-steady state conditions. We demonstrate that the O₂ dynamics in biofilms is governed by a complex interaction between biomass distribution, mass transfer and flow that cannot be directly inferred from structural information on biomass distribution alone.

Comparison of Models Describing Light Dependence of N2 Fixation in Heterocystous Cyanobacteria

ABSTRACT The abilities of four models to describe nitrogenase light-response curves were compared, using the heterocystous cyanobacterium Nodularia spumigena and a cyanobacterial bloom from the Baltic Sea as examples. All tested models gave a good fit of the data, and the rectangular hyperbola model is recommended for fitting nitrogenase-light response curves. This model describes an enzymatic process, while the others are empirical. It was possible to convert the process parameters between the four models and compare N2 fixation with photosynthesis. The physiological meanings of the process parameters are discussed and compared to those of photosynthesis.

A simple optode based method for imaging O2 distribution and dynamics in tap water biofilms

A ratiometric luminescence intensity imaging approach is presented, which enables spatial O2 measurements in biofilm reactors with transparent planar O2 optodes. Optodes consist of an O2 sensitive luminescent dye immobilized in a 1-10 μm thick polymeric layer on a transparent carrier, e.g. a glass window. The method is based on sequential imaging of the O2 dependent luminescence intensity, which are subsequently normalized with luminescent intensity images recorded under anoxic conditions. We present 2-dimensional O2 distribution images at the base of a tap water biofilm measured with the new ratiometric method and compare the results with O2 distribution images obtained in the same biofilm reactor with luminescence lifetime imaging. Using conventional digital cameras, such simple normalized luminescence intensity imaging can yield images of 2-dimensional O2 distributions with a high signal-to-noise ratio and spatial resolution comparable or even surpassing those obtained with expensive and complex luminescence lifetime imaging systems. The method can be applied to biofilm growth incubators allowing intermittent experimental shifts to anoxic conditions or in systems, in which the O2 concentration is depleted during incubation.

LIGHT ACTION SPECTRA OF N2 FIXATION BY HETEROCYSTOUS CYANOBACTERIA FROM THE BALTIC SEA1

An on‐line, laser photo‐acoustic, trace gas detection system in combination with a stepper motor‐controlled monochromator was used to record semicontinuous light action spectra of nitrogenase activity in heterocystous cyanobacteria. Action spectra were made of cultures of Nodularia spumigena Mertens ex Bornet & Flahault, Aphanizomenon flos‐aquae Ralfs ex Bornet & Flahault, and Anabaena sp. and from field samples of a cyanobacterial bloom in the Baltic Sea. Nitrogenase activity was stimulated by monochromatic light coinciding the red and blue peaks of chl a, the phycobiliproteins phycocyanin (allophycocyanin) and phycoerythrin, and several carotenoids. Because nitrogenase is confined to the heterocyst, it was concluded that all photopigments must have been present in these cells, were involved in light harvesting and photosynthesis, and supplied the energy for N2 fixation. The species investigated showed marked differences in their nitrogenase action spectra, which might be related to their specific niches and to their success in cyanobacterial blooms. Moreover, light action spectra of nitrogenase activity shifted during the day, probably as the result of changes in the phycobiliprotein content of the heterocyst relative to chl a. Action spectra of nitrogenase and changes in pigment composition are essential for the understanding of the competitive abilities of species and for the estimation of N2 fixation by a bloom of heterocystous cyanobacteria.

Counting Viruses and Bacteria in Photosynthetic Microbial Mats

ABSTRACT Viral abundances in benthic environments are the highest found in aquatic systems. Photosynthetic microbial mats represent benthic environments with high microbial activity and possibly high viral densities, yet viral abundances have not been examined in such systems. Existing extraction procedures typically used in benthic viral ecology were applied to the complex matrix of microbial mats but were found to inefficiently extract viruses. Here, we present a method for extraction and quantification of viruses from photosynthetic microbial mats using epifluorescence microscopy (EFM) and flow cytometry (FCM). A combination of EDTA addition, probe sonication, and enzyme treatment applied to a glutaraldehyde-fixed sample resulted in a substantially higher viral (5- to 33-fold) extraction efficiency and reduced background noise compared to previously published methods. Using this method, it was found that in general, intertidal photosynthetic microbial mats harbor very high viral abundances (2.8 × 1010 ± 0.3 × 1010 g−1) compared with benthic habitats (107 to 109 g−1). This procedure also showed 4.5- and 4-fold-increased efficacies of extraction of viruses and bacteria, respectively, from intertidal sediments, allowing a single method to be used for the microbial mat and underlying sediment.

Microscale spatial distributions of microbes and viruses in intertidal photosynthetic microbial mats

Intertidal photosynthetic microbial mats from the Wadden Sea island Schiermonnikoog were examined for microscale (millimetre) spatial distributions of viruses, prokaryotes and oxygenic photoautotrophs (filamentous cyanobacteria and benthic diatoms) at different times of the year. Abundances of viruses and prokaryotes were among the highest found in benthic systems (0.05–5.43 × 1010 viruses g−1 and 0.05–2.14 × 1010 prokaryotes g−1). The spatial distribution of viruses, prokaryotes and oxygenic photoautotrophs were highly heterogeneous at mm scales. The vertical distributions of both prokaryotic and viral abundances were related to the depth of the oxygenic photoautotrophic layer, implying that the photosynthetic mat fuelled the microbial processes in the underlying layer. Our data suggest that viruses could make an important component in these productive environments potentially affecting the biodiversity and nutrient cycling within the mat.

Reactive oxygen production induced by near-infrared radiation in three strains of the Chl d-containing cyanobacterium Acaryochloris marina.

Cyanobacteria in the genus Acaryochloris have largely exchanged Chl a with Chl d, enabling them to harvest near-infrared-radiation (NIR) for oxygenic photosynthesis, a biochemical pathway prone to generate reactive oxygen species (ROS). In this study, ROS production under different light conditions was quantified in three Acaryochloris strains (MBIC11017, HICR111A and the novel strain CRS) using a real-time ethylene detector in conjunction with addition of 2-keto-4-thiomethylbutyric acid, a substrate that is converted to ethylene when reacting with certain types of ROS. In all strains, NIR was found to generate less ROS than visible light (VIS). More ROS was generated if strains MBIC11017 and HICR111A were adapted to NIR and then exposed to VIS, while strain CRS demonstrated the opposite behavior. This is the very first study of ROS generation and suggests that Acaryochloris can avoid a considerable amount of light-induced stress by using NIR instead of VIS for its photosynthesis, adding further evolutionary arguments to their widespread appearance.

Disruption of photoautotrophic intertidal mats by filamentous fungi

Ring-like structures, 2.0-4.8 cm in diameter, observed in photosynthetic microbial mats on the Wadden Sea island Schiermonnikoog (the Netherlands) showed to be the result of the fungus Emericellopsis sp. degrading the photoautotrophic top layer of the mat. The mats were predominantly composed of cyanobacteria and diatoms, with large densities of bacteria and viruses both in the top photosynthetic layer and in the underlying sediment. The fungal attack cleared the photosynthetic layer; however, no significant effect of the fungal lysis on the bacterial and viral abundances could be detected. Fungal-mediated degradation of the major photoautotrophs could be reproduced by inoculation of non-infected mat with isolated Emericellopsis sp., and with an infected ring sector. Diatoms were the first re-colonizers followed closely by cyanobacteria that after about 5 days dominated the space. The study demonstrated that the fungus Emericellopsis sp. efficiently degraded a photoautotrophic microbial mat, with potential implications for mat community composition, spatial structure and productivity.